Glutamate is the principal excitatory neurotransmitter in the central nervous system of mammals. During synaptic transmission, the post-synaptic responses occur via ionotropic and metabotropic glutamate receptors. Metabotropic receptors operate via G-proteins and mobilize calcium ions from intracellular compartments. Activation of ionotropic receptors results in increase in permeability of postsynaptic membrane for sodium, potassium and calcium cations by opening a ion channel, which is an integral parts of the receptors.
Typical examples of ionotropic receptors are N-methyl D-aspartate (NMDA) receptors, AMPA and kainate receptors. Although current knowledge suggests specific role of various types of superfamily of glutamate receptors in the glutamate-induced excitotoxicity, ionotropic receptors are generally considered to be a key player in these processes. Activation of ionotropic receptors leads to alterations in intracellular concentrations, of various ions, mainly of Na+ and Ca2+. Current research demonstrates that beside calcium, elevated intracellular levels of sodium ions can also lead to neuronal death. In neuronal cultures and in retina the activation of glutamate receptors may lead to damage even by sodium cations in absence of extracellular calcium ions. Nonetheless, toxicity of elevated glutamate levels is usually associated with elevations in intracellular concentrations of Ca2+. Currently it is well established that there is a direct relationship between excessive influx of calcium into cells and glutamate-induced damage to neurons. Glutamate-induced pathological calcium elevation is usually ascribed to prolonged activation of ionotropic receptors. Elevation in intracellular calcium then may trigger the down-stream neurotoxicity cascade, which involves uncoupling of mitochondrial electron transport from ATP production, supranormal activation of enzymes such as calpain and other proteases, induction of specific protein kinases, NO-synthase, calcineurins and endonucleases. These changes may also promote the production of toxic reactive molecules such as reactive oxygen species (ROS) and induce changes in cytoskeleton architecture and activation of signals leading to apoptosis and mitochondrial damage (Villmann and Becker, 2007).
A number of preclinical studies show a remarkable ability of NMDA receptor antagonists to prevent from the excessive exocytose of glutamate and damage to the CNS. From the clinical point of view; however, their therapeutic potential is rather limited. Regarding the fact that glutamate receptors are ones of the most abundant in the CNS, application of their antagonists leads to wide variety of side effects, ranging from motor impairment to induction of psychotic symptoms. On the contrary, a large divergence of NMDA receptors and differences in their distribution at synapses and at extrasynaptic sites offer a possibility to search for drugs which selectively influence only a limited subset of NMDA receptors and thus to avoid the induction of unexpected side effects, while retaining their therapeutic neuroprotective activity.
Previous results demonstrated that naturally occurring 3α5β-pregnanolone sulfate affects the activity of NMDA receptor by a use-dependent manner. As a consequence this molecule has a more pronounced inhibitory action on the tonically active NMDA receptors than on those phasically activated by glutamate during synaptic transmission. It was also demonstrated that activation of extrasynaptic tonically activated NMDA receptors is very important for excitotoxic action of glutamate (Petrovic et al., 2005).
Therefore, we have started the development and testing of novel NMDA receptor antagonists derived from neurosteroids. These newly synthesized drugs exhibit affinity for extrasynaptic NMDA receptors. What is more important, previous electrophysiological studies showed that these compounds bound preferentially to open NMDA receptor channels. Our compounds lack affinity for other types of receptor; it is thus presumed that they will not affect signal transmission between neurons. The suggested mechanisms of their action are the blockade of extrasynaptic tonically activated NMDA receptors and prevention of excessive action of glutamate on neurons.
In the last decade, the biomedical research focused on the study of the role of neurosteroids in the pathogenesis of number of neuropsychiatric diseases and evaluation of their therapeutic potential. Mechanisms of action of neurosteroids are conventionally associated with their activity on NMDA and GABA-A receptors. A number of experimental studies with animal models show their potential in therapy of several diseases of CNS, including neurodegenerative disorders, multiple sclerosis, affective disorders, alcoholism, pain, insomnia or schizophrenia (Morrow, 2007; Weaver, 2000).
Neurosteroids also play a crucial role in the regulation of reactivity to stress and stress-related CNS disorders. Corticosteroid levels are known to acutely increase after exposition to a stressor; this represents an adaptive mechanism. On the other hand, experimental models of chronic stress and depression in laboratory rodents show decreased levels of neurosteroids both in brain and plasma. Similar findings are often reported in patients suffering from depressions and pre-menstruation syndrome suggesting impairments in the CNS homeostatic mechanisms in stress-related neuropsychiatric disorders.
Steroid compounds affect activity and plasticity of neural and glial cells during early in life, and later in development they play an essential trophic and neuroprotective role in the adult CNS. Steroids are released by sexual and adrenal glands as well as in the CNS. Steroids secreted by peripheral glands reach brain, medulla and spinal cord via blood circulation. Nonetheless, some neural steroids (i.e., neurosteroids) are synthesized directly in the CNS. The most studied neurosteroids are represented by pregnenolone, progesterone, dehydroepiandrosterone (DHEA) and their reduced metabolites and sulphate esters. Not much is known about regulation of neurosteroid synthesis in the CNS, but it is generally assumed that they may underlie interaction of multiple cell types in the CNS. For example, synthesis of progesterone by Schwann cells surrounding peripheral nerves is regulated by signals diffusing from neurons.
Neurotrophic and neuroprotective properties of some neurosteroids were convincingly demonstrated both in cultures and in vivo. Progesterone plays a pivotal role in neurological recovery from traumatic brain and spinal cod injury by mechanisms including protection against excitotoxic damage to the brain, lipid peroxidation and by induction expression of specific enzymes. For example, after cutting the spinal cord, this steroid increases the number of NO-synthase-expressing astrocytes in place adjacent to cut both in the distal and proximal segment of the cord.
This steroid was also shown to regulate formation of new myelin sheaths. This fact was shown in regenerating rat sciatic nerve in the culture with sensory neurons and Schwann cells. Progesterone also supports myelination by activation of genes coding for proteins participating in this process.
As mentioned before, neurosteroids importantly modulate the function of membrane receptors for various neurotransmitters, namely GABAA receptors, NMDA receptors and sigmal-opioid receptors. These mechanisms are most likely responsible for psychopharmacological effects of steroids and may at least partly account for their anticonvulsant, anxiolytic, neuroprotective and sedation effects as well as for their influence upon learning and memory functions. For instance, pregnanolone sulphate was shown to be capable of reversing cognitive deficit in aged animals and exerting a protective effect on memory in several amnesia models. Recent studies have demonstrated direct effect of neurosteroids on intracellular receptors. Despite absence of direct evidence for binding of neurosteroids to corticoid receptors, they may obviously modulate their function indirectly, by interaction with protein kinases C and A, MAP-kinase (MAPK) or CaMKII. Moreover, pregnanolone and pregnanolone sulphate were shown to affect microtubule-associated proteins and increase the rate of microtubule polymeration, which may in turn affect neuronal plasticity. We are far from fully understanding these newly-described effects of neurosteroids, however, their potential role in neuroprotective mechanisms deserves scientific attention.
Sulfated esters of neurosteroids also play a physiological role in the regulation of receptors for excitatory and inhibitory neurotransmitters and participate in the natural protective properties of CNS tissue. Sulphated esters of neurosteroids and their analogues are promising molecules, potentially beneficial for treatment of CNS disorders. Nonetheless, a ratio between neurosteroids and their sulfated esters is maintained enzymatically in the CNS tissue in vivo. Exogenous administration of sulfated esters may not lead to improvement in the protective functions due to increased enzyme activity in the CNS converting them to inactive forms. The invented molecules are metabolically stable analogues of sulfated esters of neurosteroids; moreover, they pass the blood-brain barrier more readily due to their chemical structure. Sulfated and thus polar steroids compounds generally penetrate the blood-brain barrier with difficulty, but it was demonstrated that intravenously administered pregnanolone sulphate can reach the brain. This transport of sulphated analogs is probably mediated by active exchange mechanisms associated with so-called organic anion transport protein (OATP), which is expressed in the cells throughout the CNS.
Advantage of our molecules is that they retain similar pharmacological and physiological properties as pregnanolone sulphate, but they are not degraded by sulfatases into non-conjugated metabolites.